Immunotherapy has raised great interest in the therapy of substantially every disease which can be targeted to an undesired antigen, for example a pathogen, tumor antigen, growth factors or pathologic protein aggregates. On the other hand, antibody responses in a subject may be the cause of a disease, for example in autoimmune and some inflammatory disorders, where autoantibodies for some reason attack endogenous proteins, cells and tissue.
In the past decades several technologies have been developed to isolate monoclonal antibodies and to produce humanized or fully human antibodies; see, e.g., references cited in international application WO 2007/068758 also granted as European patent EP 1 974 020 B1, in particular in sections [0002] to [0027], the disclosure content of which is incorporated herein by reference.
Typically, the isolation of antibodies, for example monoclonal antibodies, from B cells relies on cloning and expression of the immunoglobulin genes. This can be done by using phage display libraries of scramble VH and VL genes from B cells, or by isolation of paired VH and VL genes from single B cells using single cell PCR or from immortalized B cell clones.
Hitherto, the prior art was aiming at providing methods for establishing more optimized processes in which a high-through-put analysis of the secreted antibodies can be performed on the largest possible population of immortalized antibody-secreting cell lines maintained in cell culture conditions. In this context, several means and methods were investigated in order to enhance the efficiency of EBV immortalization and of cellular cloning of EBV-immortalized cells; see, e.g., international applications WO 2004/076677 and WO 2007/068758.
Almost all methods for obtaining immortalized antibody secreting cells have been established with B lymphocytes from healthy donors or infectious disease patients, which showed reactivity with viral antigens; see, e.g., Traggiai et al., Nat. Med. 10 (2004), 871-875 and Funaro et al., BMC Biotechnology 8 (2008), 85.
However, the current methods may not be as efficient for the isolation of antigen-independent memory B cells from patients experiencing an aberrant immune response, for example in autoimmune disorders wherein subsets of B and T lymphocytes become anergic, prone to apoptosis or otherwise dysregulated. For example, distinct mature B-cell subsets that accumulate with age, termed age-associated B cells have been discovered in mice, which are uniquely responsive to innate stimuli and refractory to CD40 and BCR stimulation; see, e.g., Hao et al., Blood 118 (2011), 1294-1304. They appear to be generated from mature B cells that exhaustively expand during the individual's lifetime and thus may represent an “exhausted” lymphocyte cell population. Moreover, an IgD− CD27− population, possibly corresponding to exhausted memory B cells accumulates in elderly humans; see, e.g., Colonna-Romano et al., Mech. Ageing Dev. 130 (2009), 681-690. Furthermore, the immortalization efficiency of memory B cells from severely infected patients such as HIV-1 patients which typically also suffer from opportunistic diseases was reported to be significantly lower than that of non-HIV-1 infected donors. This finding was discussed to be due to the presence of “exhausted” memory B cells, characterized by the expression of inhibitory receptors and low levels of CD21; see, e.g., Moir et al., Nat. Rev. Immunol. 9 (2009), 235-245.
For the same reasons, phenotypically distinct populations of autoreactive B cells that have become functionally limited upon stimulation and/or display reduced viability in cell culture conditions may be found at increased frequency in autoimmune and some inflammatory diseases. It has been shown that self-Antigen (Ag) recognition results in severely diminished IgG response (Detanico et al., J. Immunol. 189 (2012), 4275-4283; Chumley et al., J. Immunol. 169 (2002), 1735-1743). Autoreactive B cells that can be found in patients with autoimmune and some inflammatory diseases are likely to be continually exposed to antigens and therefore anergic (Gauld et al, Nat. Immunol. 6 (2005), 1160-1167). These cells are expected to be functionally limited upon stimulation and/or display reduced viability in cell culture conditions.
Therefore, memory B cells from patients suffering from for example autoimmune or inflammatory diseases, in particular characterized by disregulated memory B cells or exhausted and less viable B cells, respectively, may not be sufficiently amenable to current immortalization protocols. However, such patients because of their immunoglobulin repertoire matured during the course of their disease may provide a valuable source of human antibodies against various therapeutic targets.
Accordingly, it would be highly desirable to provide methods for establishing processes in which, by applying specific means and conditions in cell culture for improving the viability and level of immunoglobulin production sufficient for the characterization of the immunoglobulin secreted B cells and cloning of corresponding immunoglobulin gene repertoire.
This problem has been solved by the embodiments characterized in the claims and following below as illustrated in the Examples and Figures.